This invention relates to an optical sensing unit. More particularly, it relates to a luminescence-based chemical and biochemical optical sensing unit and to the uses thereof.
In human diagnostics, an increasing demand for the detection of extremely low concentrations of biochemically relevant molecules in small sample volumes has triggered research efforts towards more sensitive and selective sensors. Optical sensors are favored because of their chemical stability and ease of fabrication. Bioaffinity sensors based on luminescence excitation schemes combine (bio-)chemical selectivity due to the application of recognition elements specifically binding the analyte molecules, with spatial selectivity originating from evanescent field excitation techniques. Common to the various evanescent field sensing methods that have been developed, interaction with the analyte molecules is restricted to the penetration depth of the evanescent field, thus emphasizing the processes occurring at the sensing surface or within the sensing layer and discriminating processes in the bulk medium.
The combination of fiber-shaped evanescent field sensors with bioaffinity assays, using fluorescent tracer probes for signal generation, has proven capability and is widely used. A detection limit of 7.5xc3x9710xe2x88x9214 M fluorescein-labeled complimentary DNA in a DNA hybridization assay using multimode fibers has been reported. On the other hand, in recent years evanescent field sensors with planar transducer geometries have been adapted for the detection of biomolecules using the principle of effective refractive index changes such as surface plasmon resonance, grating couplers, and interferometers. They are associated with the attractive feature of direct sensing, without the necessity of using any labels. However, the signals of these devices are directly associated with the adsorbed molecular mass which limits the sensitivity of these configurations. Typically, detected concentrations hardly range below 10xe2x88x9210 M.
To match the goal of extremely low detection limits in demand by gene probe analysis as well as the diagnostics of diseases and infections, it has been proposed to use single-mode metal oxide waveguides as transducers for luminescence-based bioaffinity sensors. This transducer geometry offers advantages due to ease of production of planar chips, sensor handling, increased excitation efficiency of the luminescence labels, and fluid handling of minute sample volumes. The features of a planar evanescent field transducer for a luminescence detection scheme and the design of a sensor system based on such waveguides are described in a paper by D. Neuschxc3xa4fer et al, entitled xe2x80x9cPlanar waveguides as efficient transducers for bioaffinity sensorsxe2x80x9d, Proc. SPIE, Vol. 2836 (1996). The sensor described uses a single-mode planar waveguide consisting of a tantalum pentoxide waveguiding film deposited on a glass substrate. For luminescence detection, in general, a xe2x80x9cvolume detectionxe2x80x9d configuration shown in FIG. 1 is used. In this case, the bottom half-sphere part of the luminescence light, which is excited by the evanescent field and then isotropically emitted, is collected underneath the sensor chip, using a high numerical aperture lens or lens system. Two identical interference filters are used for discrimination of excitation light. Signal detection is performed using either photodiodes in combination with high-gain amplifiers, or a selected photomultiplier in combination with a photon-counting unit. As an alternative, the part of the luminescence signal which is coupled back into the waveguiding film may be collected using a second, outcoupling grating (not shown). This is known as xe2x80x9cgrating detectionxe2x80x9d. The angular separation of outcoupled light of different wavelengths offers the additional feature of simultaneous determination of the- transmitted excitation and the emitted luminescence light intensities, It is also possible to combine the two methods in one device to provide simultaneous xe2x80x9cvolumexe2x80x9d and xe2x80x9cgratingxe2x80x9d detection. A detailed comparison of the two methods is discussed by G. L. Duveneck et al, in a paper entitled xe2x80x9cA novel generation of luminescence-based biosensors: single-mode planar waveguide sensorsxe2x80x9d, Proc. SPIE, Vol. 2928 (1996).
In the present application, the term xe2x80x9cmeasurement fieldxe2x80x9d refers to the smallest area of a sensor field capable of discrimination by a photoelectric detector used to detect luminescence. The present invention addresses the need for simultaneous, spatially selective excitation and highly sensitive detection of luminescence signals from an array of measurement fields. Conventional bioaffinity sensors typically rely upon macroscopic imaging of emitted luminescence from a single, large measurement field. A direct transfer of this technique to arrays of measurement fields suffers from inherent optical crosstalk and optical pick-up of background radiation to the extent that the detection limit is often not sufficiently low for many applications. Furthermore, when macroscope optical elements are used to provide a degree of lateral resolution, the distance between the measurement field and the detector array needs to be quite substantial, thereby increasing the overall size of the system. For luminescence detection in extremely small measurement fields and volumes, confocal laser fluorescence microscopy is a very sensitive method. The detection of individual molecules has been demonstrated with excitation areas as small as the diffraction-limited focus of the laser excitation light, i.e. of the order of one wavelength. However, the excitation and detection of a large number of measurement cells in an array requires a lateral translation of the sample with respect to the measuring arrangement to allow sequential measurement of each measurement cell in the array. Accordingly, the time required to receive the signals of a substantial array of measurement fields is prolonged and the relative cost of this type of system itself is expensive due to the size and complexity of the instrument.
According to a first aspect of the present invention, there is provided an optical sensing unit which comprises at least one sample measurement cell, at least one excitation light source acting upon the or each measurement cell to provide one or more sensor fields defining an array of measurement fields, a photoelectric detector array for detecting the intensity of light emitted from the or each measurement cell in response to excitation light, and an array of waveguides or channels for directing light emitted from each measurement field to a respective portion of the photoelectric detector array, characterized in that the array wave-guides or channels have separate beam guiding of the excitation light and emission light for each waveguide or channel.
The present invention addresses the need to improve the ratio of a detected luminescence signal to background xe2x80x9cnoisexe2x80x9d in a luminescence based measurement method. It achieves this by providing a form of beam guiding for light emitted from an array of relatively small measurement fields associated with a number of sensing fields to eliminate optical cross-talk usually associated with conventional macroscopic imaging of adjacent measurement fields. In the preferred examples, an array of waveguides or channels are used with separate beam guiding of the excitation light and emission light for each waveguide or channel.
In one preferred example of the present invention, the one or more sensor fields are provided by the use of a number of planar evanescent field transducers. Preferably, the sensor fields comprise a number of spaced apart optical waveguides, which are preferably arranged in parallel segments. The excitation light is coupled into the array of optical waveguides to establish a number of spatially separated evanescent sensor fields.
Preferably, the or each waveguide is a single-mode metal oxide planar transducer. Where an array of waveguides is provided, they may be integrated on a single substrate. In a preferred embodiment, the spatially separated evanescent sensor fields are established by contacting a continuous metal oxide waveguiding layer, deposited over the whole substrate, with a structured, absorbing surface, having a geometrical arrangement corresponding to the number and pitch of the sensor fields.
The or each measurement cell acts as a conduit or well for a liquid sample. In one preferred example of the present invention, a flow cell housing is provided which is partitioned to form an array of fluid channels having a geometrical arrangement corresponding to the number and pitch of the sensor fields. A preferred form of flow cell is the so-called counter-current flow cell described in the Proceedings on the xcexc-TAS ""96 in Basel, Special Issue 1996 by Analytical Methods and Instrumentation, pp. 158-162. This particular flow cell is adapted to cooperate with a planar transducer substrate to form a fluid-tight compartment.
Preferably, the optical sensing unit comprises a number of optical coupling elements for coupling excitation light into the measurement cells. Preferably, the coupling elements comprise a number of diffraction gratings. The optical sensing unit may also include optical coupling elements for coupling light out of the measurement cells. In particular, in a planar evanescent field transducer configured for grating detection, a second diffraction grating is used to couple out back-coupled luminescence onto the detector array. Alternatively, or in addition, a second diffraction grating can be used to couple out transmitted excitation light onto another detector for reference purposes.
Although a plurality of excitation light sources may be used, one for each measurement cell, preferably a single excitation light source is provided which, if necessary, is multiplexed onto the in-coupling elements to provide an array of parallel light beams. A preferred form of optical multiplexer is a Dammann diffraction grating which, in combination with a suitable optical element such as a lens, is used to provide a parallel set of light beams for establishing a number of sensor fields. Other suitable optical multiplexers include reflection gratings, refractive elements, microlens arrays, microprism arrays, and Fresnel lens arrays.
A laser is a suitable excitation light source, with the appropriate wavelength selected, whether in the form of a laser diode, a solid state laser, or a gas laser. Further, one or more sets of optical shutters may be provided selectively to block excitation light to a number of the measurement cells. Furthermore, the excitation light path may be folded to reduce the size of the optical sensor.
In another preferred example of the present invention, the excitation light beams are not coupled into the waveguiding layer, but launched onto the measurement fields in a configuration similar to classical surface or transmissive illumination. In this example, the amount of luminescence light that is excited in the nearfield of the waveguide (i.e. within about one wavelength from the waveguide surface), is partially coupled into the waveguiding layer and is outcoupled using optical coupling elements, such as diffractive gratings, located adjacent to the measurement fields. One or more arrays of apertures and of microlenses may be provided in the optical path between the outcoupling gratings and the corresponding photoelectric detector array. Additionally, interference filters or an array of interference filters may be arranged in the optical detection path for further discrimination of excitation light.
In yet another preferred example of the present invention, the excitation and detection area or volume of the measurement fields are identical and preferably arranged in a multiplexed configuration of a confocal optical excitation and detection system, with a separate confocal excitation and detection path for each measurement field.
In a further preferred embodiment of the present invention, the multiplexed confocal optical excitation and detection system is constructed around a multiplexed planar capillary chromatography chip, or a planar capillary array electrophoresis chip, with separate excitation and detection for each separation capillary.
In the optical detection arrangement of the present invention, the size and pitch of each measurement field may correspond to the size and pitch of the photoelectric detector array. However, in practice the size of each measurement field is typically somewhat larger than the size of the respective portion of the detector array allocated to it. In the latter case, light emitted from each measurement field is focused onto the respective portion of the detector array.
Preferably, in a xe2x80x9cvolume detection configuration,xe2x80x9d the photoelectric detector array is located immediately above or below the measurement cell so as to pick up a component of luminescence light which is radiated isotropically from the measurement fields into space.
Preferably, the optical sensing unit comprises an array of microlenses, which are arranged between the plane of the measurement cell and that of the detector array, to focus light emitted from each measurement field onto a respective portion of the detector array. The array of microlenses may advantageously be integrated with a substrate of the optical sensing unit.
Preferably, the optical detector further comprises an array of apertures adapted to the geometry of the measurement fields to provide optical isolation of emitted light between adjacent measurement fields. The apertures may be substantially planar or may extend orthogonally to the plane of the detector array.
Preferably, the optical sensor further comprises an optical filter or array of optical filters located in a plane between the measurement fields and the detector array to provide signal discrimination.
Examples of suitable photoelectric detectors include multi-channel photomultipliers, CCD arrays, CCD cameras and CMOS devices.
According to a second aspect of the present invention, there is provided a process for determining an analyte by using the present optical sensing unit which comprises introducing a liquid sample into the at least one sample measurement cell, directing a source of excitation light into the at least one measurement cell to establish one or more sensor fields defining an array of measurement fields, guiding luminescence light emitted from each of the measurement fields to a respective portion of the detector array, and detecting the intensity of light emitted by each measurement field, characterized in that the means comprise an array of waveguides or channels with separate beam guiding of the excitation light and emission light for each waveguide or channel.
Preferably, the process comprises the step of directing light into the at least one measurement cell to establish an array of sensor fields.
As will be appreciated by those skilled in the art, the present optical sensor finds a wide variety of applications among those known in the field. Preferred examples include optical determinations of absorbed or excited light in miniaturized and multiplexed chromatographic separation arrays, capillary electrophoresis arrays, or arrays of chemical or biochemical sensors.
The optical sensor according to the present invention is especially suitable for the quantitative determination of biochemical substances in a bioaffinity sensor system. It may be used to test samples as diverse as egg yolk, blood, serum, plasma, lymph, urine, surface water, soil or plant extracts, and a bio- or synthesis process or both. It may also be used, for example, in the quantitative or qualitative determination of antibodies or antigens, receptors or their ligands, oligonucleotides, DNA or RNA strains, DNA or RNA analogues, enzymes, enzyme substrates, enzyme cofactors or inhibitors, lectins and/or carbohydrates.